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Abstract

Sleep is the most basic and essential physiological requirement for mental health, and sleep disorders pose potential risks of metabolic and neurodegenerative diseases. Tryptic hydrolysate of αS1-casein (αS1-CH) has been shown to possess stress relieving and sleep promoting effects. However, the differential effects of αS1-CH on electroencephalographic wave patterns and its effects on the protein levels of γ-aminobutyric acid A (GABAA) receptor subtypes in hypothalamic neurons are not well understood. We found αS1-CH (120, 240 mg/kg) increased sleep duration in mice and reduced sleep-wake cycle numbers in rats. While αS1-CH (300 mg/kg) increased total sleeping time in rats, it significantly decreased wakefulness. In addition, electroencephalographic theta (θ) power densities were increased whereas alpha (α) power densities were decreased by αS1-CH (300 mg/kg) during sleep-wake cycles. Furthermore, protein expressions of GABAA receptor β1 subtypes were elevated in rat hypothalamus by αS1-CH. These results suggest αS1-CH, through GABAA receptor modulation, might be useful for treating sleep disorders.

Sleep disorders not only reduce quality of life but also serve as risk factors of dementia (Mishima, 2016) and metabolic diseases, like atherosclerosis (Tobaldini et al., 2017), and hence, early intervention is clinically relevant as it potentially mitigates harmful consequences. Recently developed drugs that have been used to treat insomnia, but can have undesirable side effects (Kay-Stacey & Attarian, 2016). Furthermore, reports indicate lotus leaf extract augments hypnosis by binding to γ-aminobutyric acid A (GABAA) receptor (Tian and Liu, 2015; Yan et al., 2015), and that consuming dairy products supports sleep in a better way (Kitano et al., 2014). Alpha (α)s1-casein hydrolysate (αS1-CH) is a milk protein with reported chronic stress relieving properties (Guesdon et al., 2006; Kim et al., 2007). However, although the tryptic hydrolysate of αS1-casein appears to improve sleep quality (Dela Peña et al., 2016), little data is available on the way it affects pentobarbital-induced sleep in mice or influences EEG band rhythms during stages of sleep. Furthermore, it has not been determined whether αS1-CH mediates its hypnotic action in mice via GABAA receptor in hypothalamus. Therefore, we investigated the effects of αS1-CH on sleep duration, sleep quality as determined by electroencephalography, and on the protein expression of GABAA receptor subunits (α1, β1, γ3) in the rat hypothalamus.

Animals and treatments

Male C57BL/6 mice (28–30 g) and Sprague-Dawley rats (male, 260–280 g) were purchased from the Orient Bio (Seoul, Korea) and allowed free access to water and food. Mice were grouped into six per cage, and maintained in an ambient atmosphere at 23°C under a 12 h diurnal light cycle. Rodents were divided into groups: 6 groups of mice for sleep testing, 3 groups of rats for EEG recording, and 3 groups of rats for western blotting. All behavioral experiments were carried out in a nearby room maintained where under the same environmental conditions. Experiments were conducted according to Animal Care and Use Guidelines of the School of Medicine, Ewha Womans University, Korea.

Mice were given a single dose (30–240 mg/kg, p.o.) of αS1-CH or saline 30 min prior to an injection of pentobarbital sodium (42 mg/kg, i.p.) to determine the onset and duration of sleep, as previously described by Ma et al. (2009) with slight modification. Time elapsed between disappearance (sleep onset) and reappearance of righting reflex (up to a maximum of 2 h) was defined as sleep duration. Experiments were performed in mouse cages with aspen bedding. Animals that did not sleep within 15 min after pentobarbital injection were excluded. Rats were treated with αS1-CH (150 or 300 mg/kg) orally once per day for 3 days before electroencephalography (EEG) or western blotting. EEG recordings were started at 2 hrs after last treatment, and hypothalami were collected at 6 hrs after last treatment for western blotting.

Electroencephalography

Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and a transmitter was implanted for EEG recording via telemetry as previously described (Sanford et al., 2006). Briefly, in each case, the body of the transmitter was subcutaneously implanted just posterior to the scapula using three sutures for stabilization. The transmitter electrodes were led subcutaneously to the skull, and their bare ends were placed in contact with the dura through holes in the skull. Electrodes were anchored to the skull with screws and dental cement. All surgical procedures were performed stereotaxically under aseptic conditions.

For telemetric recording of cortical EEG signals, transmitter gain was set at −0.5/+0.5 volts per unit. Raw output signals, which ranged from 0.5 to 0.0 Hz, were processed using a Data Sciences analog converter and routed to an analog-to-digital (AD) converter (Eagle PC30, Data Sciences International, St. Paul, MN, USA), which digitized EEG and activity signals. Subsequently data were transferred to a computer and graphically displayed. An on-line fast Fourier transformation (FFT) program was used to analyze EEG data and generate power density values from 0.0 to 20.0 Hz at a resolution of 0.5 Hz. FFT data were further averaged between 0 to 20 Hz at 10-s intervals. Sleep data and FFT results were saved to hard disk every 10 s for additional off-line analysis. Number of animal movements related to telemetry receiver generated transistor-transistor logic pulses were viewed as measures of activity. Data were gathered on the 1st and 3rd days after αS1-CH treatment and percentage power densities were calculated. EEG signals were measured for 6 hrs between 11:00 am 5:00 pm. Each group contained 5–6 rats.

Statistical analysis

Sleep onset and duration data were analyzed using analysis of variance (ANOVA). The Newman-Keuls test was used to perform intergroup comparisons. Values were expressed as means ± SEM, and statistical significance was accepted for p values <0.05.

RESULTS

Pretreatment with αS1-CH prolonged sleep duration in mice

It has been demonstrated αS1-casein hydrolysate protects rat from chronic mild stress-induced sleep disorders (Guesdon et al., 2006). In our preliminary experiment, αS1-CH showed an anxiolytic effect at relatively low doses (25, 50 mg/kg) in mice (data not shown). Therefore, we investigated whether αS1-CH improves sleep duration in pentobarbital-treated mice. Mice treated with αS1-CH in the dose range 30–240 mg/kg tended to have lower sleep-onset times (Fig. 1A). However, this effect of αS1-CH was not significant as compared with pentobarbital controls. Diazepam (1 mg/kg, i.p.) exhibited significantly earlier sleep-onset times than that of control after pentobarbital treatment. One-way ANOVA showed significant differences in sleep induction between control and diazepam group [F (5,50)=5.0, p<0.01]. In contrast, the duration of sleep significantly elevated when mice were treated with αS1-CH at higher doses (120 or 240 mg/kg, p.o.) [F (5,51)=15.02, p<0.01] (Fig. 1B).

Rats pretreated with αS1-CH had fewer sleep-wake cycles

Sleep-wake cycle disruption has been associated with stress, which suggests that reducing the number of sleep-wake cycles may provide relief from neurodegenerative diseases (Cedernaes et al., 2017). Therefore, we investigated whether αS1-CH could reduce the number of sleep-wake cycle disruptions in rats. In the preliminary test, administration of αS1-CH at doses of 50 or 100 mg/kg did not significant affect sleep/wake cycles or EEG patterns in rats, and thus, the dose of αS1-CH was increased to 150 or 300 mg/kg. We found αS1-CH at 300 mg/kg significantly reduced the number of sleep-wake cycles by ∼50% (Fig. 2). Furthermore, total time awake was reduced by αS1-CH pretreatment and total asleep was increased (Fig. 3). Although REM sleep was decreased and NREM sleep was increased after treatment with αS1-CH, no significant differences were found between treatment groups (Fig. 3).

The effect of αS1-CH on frequency bands of EEG during sleep-wake cycles

Protein αS1-CH (150 or 300 mg/kg, p.o.) was administered to rats once per day for 3 days. Wakefulness, REM sleep and NREM sleep were monitored using the power densities of delta (δ), theta (θ), and alpha (α) frequency bands in rats treated without or with αS1-CH (150 or 300 mg/kg). Whereas the percentage of θ power density was significantly increased by treatment with αS1-CH (300mg/kg) in sleep-wake cycles, δ frequency bands showed negligible differences. On the other hand, treatment with αS1-CH at 300 mg/kg decreased the percentage of α power density (Fig. 4).

αS1-CH modulated the expression levels of β1 and γ3 subtypes of GABAA receptor in the rat hypothalamus

GABAA receptor subtypes in neuronal tissue have been reported to be targets for insomnia treatment (Luppi et al., 2017). We investigated whether the protein expressions of the GABAA receptor subunits α1, β1, and γ3 were modulated in the hypothalami of rats treated with αS1-CH. Treatment using αS1-CH at 150 mg/kg or 300 mg/kg increased the protein expression of β1, but the protein expression level of α1 and glutamic acid decarboxylase (GAD65/67; catalyzes the formation of GABA in neuronal tissues) were unaltered (Fig. 5). Although the level of γ3 tended to be elevated by αS1-CH treatment, this was not statistically significant.

DISCUSSION

Considering the importance of nutrition-based hypnosis over that of recently developed drugs with undesired side effects, our report on αS1-CH has merit with respect to prolonged duration of sleep, and fewer sleep-wake cycles, which suggests a new avenue for developing alternative therapeutic options against the insomnia experienced during stressful conditions. Sleep disorders are associated not only with mental problems (Yu et al., 2013) but are also linked to various health conditions, such as, metabolic disease and reduced testoster-one levels accompanied by altered sexual behavior (Alvarenga et al., 2015). Recently, a disordered protein architecture of receptors was suggested to be related to sleep problems (Tou and Chen, 2014), which implies a complex mechanism underlies insomnia. Several drugs that ameliorate insomnia have been developed, but many are associated with unwanted side effects (Kripke, 2016; Sirdifield et al., 2017). Alternative options have been sought, such as, acupuncture (Lee and Lim, 2016) and the use of natural products (Shi et al., 2014). Milk contains a wide variety of bioactive peptides, including those in tryptic hydrolysate of αS1-casein, which has been reported to modulate the architecture of sleep (Dela Peña et al., 2016).

Little is known of EEG band rhythms and membrane receptor expressions in hypothalamic neurons, and EEG parameters are considered essential during sleep examinations and are used to evaluate sleep patterns or problems. Therefore, we investigated the effects of αS1-casein in mouse model of pentobarbital-induced sleep.

We found αS1-CH (30–240 mg/kg) did not modulate sleep onset, but that at 120 or 240 mg/kg it prolonged sleep duration in mice. Similarly, αS1-CH at 300 mg/kg reduced the number of sleep-wake counts nearly by a half in rats. Moreover, total sleeping time was increased but wakefulness was diminished by αS1-CH at 300 mg/kg. Together, these findings strongly support previous findings that suggested the tryptic hydrolysate of αS1-casein had sleep promoting properties. Pena et al. also showed that EEG δ waves increased in NREM sleep whereas α waves decreased (Dela Peña et al., 2016). In the present study, we also found the power density of θ waves were significantly increased and α densities significantly decreased by αS1-CH (300 mg/kg) in rats. It has been known δ waves are slow waves related to the governance of sleep, and that α waves are high frequency waves related to sedatives and hypnotics (Stahl, 2008). Interestingly, it was reported that δ rhythm is predominantly seen during NREM sleep in contrast to θ rhythm, which is usually observed during REM sleep (Luppi et al., 2017). In general, in our EEG signals, θ waves were significantly enhanced during REM sleep, NREM sleep, and wakefulness when rats were treated with αS1-CH (300 mg/kg), which indicates higher concentrations of αS1-CH influence EEG signals. In contrast to θ rhythms, α rhythms are present during waking (Doroshenkov et al., 2007), and in the present study were found to be decreased by pretreatment with αS1-CH at higher concentration (300 mg/kg). This result may seem contradictory given the aforementioned EEG patterns of REM and NREM, and we cannot provide an explanation for this result. However, Rajaratnam et al. showed that melatonin administration does not significantly change δ or α activities in man (Rajaratnam et al., 2004), and suggested melatonin facilitates rather than induces sleep. This might also be the case for αS1-CH.

Despite controversies regarding the properties, functions, and subunit arrangements of GABAA receptors, they have been established to be pentameric ligand-gated channels that negatively mediate neurotransmission in the central nervous system, (Puthenkalam et al., 2016; Wongsamitkul et al., 2016). Dela Peña et al. (2016) suggested GABAA receptor subunits play a role in mediating αS1-CH induced sleeping based on results obtained using bicuculline, a competitive GABAA receptor antagonist, and that the dose-dependent increase in chloride ion influx induced by αS1-CH in cultured human neuroblastoma cells was blocked by bicuculline. In the present study, we found the protein expression of the β1 receptor subunit of GABAA was increased in the hypothalami of rats treated with αS1-CH (150, 300 mg/kg), but that α1 and GAD65/67 protein levels were unchanged. The activation of GAD plays an important role in the GABAergic system because GABA is generated from glutamate by the action of GAD. In the present study, protein levels of GAD65/67 were unaltered by αS1-CH administration, suggesting αS1-CH might not modulate GABA generation. Nevertheless, our report indicates the importance of β1 subunits of GABAA receptor in αS1-CH enhanced sleep, though further studies are warranted on receptor subtypes and their arrangements in GABAA receptor (Mohler et al., 2005; Wongsamitkul et al., 2016), especially since Liang and Marks (2014) observed the involvement of the GABAA γ2 receptor subunit in REM sleep.

In the present study, we found αS1-CH significantly enhanced pentobarbital-induced sleep duration in mice, increased total sleep, and EEG θ wave during sleep in rats. Given increased protein expressions of GABAA receptor β1 subunits after αS1-CH treatment observed in rats, further work is required to explore other GABAA receptor subtypes and their arrangements to clearly delineate the sleep-enhancing effect of αS1-CH. Nonetheless, our findings suggest αS1-CH dietary supplementation could be deployed to treat sleep disorders.

ACKNOWLEDGEMENTS

This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Science, ICT & Future Planning (MRC, 2010-0029355) and by the Chungbuk Bio International R&D Project (2014-1-01).

Figures

Fig. 1. Effects of αs1-CH on the onset and duration of sleep in pentobarbital-treated mice. Mice were food-deprived for 12 h prior to being treated with αs1-CH (30–240 mg/kg) or diazepam (DZP, 1mg/kg, i.p.). Sleep latency (A) and total sleeping time (B) were recorded for 120 min after injecting pentobarbital (42 mg/kg, i.p). Columns contain mean values and SEMs (n=8–10) as determined by ANOVA. Comparisons were made using the Newman-Keuls test. *p<0.05, **p-values of <0.01 were considered significant as compared with pentobarbital-treated controls.

Fig. 2. Effects of αs1-CH on sleep-wake counts. αs1-CH (150, 300 mg/kg) was orally administered to rats once daily for 3 days. Sleep-wake cycles were measured by EEG for 6 hrs and analyzed using Sleep Sign 2.1 software. Values were compared using the Newman-Keuls test. Column contain mean values and SEMs (n=5–6). *p-values of <0.05 were considered significant as compared with naïve controls.

Fig. 3. Differential effects of αs1-CH on sleeping stages in rats. Wakefulness, total sleep, REM sleep, and NREM sleep were determined after administering αs1-CH (150, 300 mg/kg) orally to rats once daily for 3 days. Sleeping was determined using telemetric cortical EEG records and analyzed using Sleep Sign2.1 software. To compare each group versus naïve control, we used the Newman-Keuls test. Columns represent mean values and SEMs (n=5–6). *p<0.05 compared with naïve controls.

Fig. 4. The effects of αs1-CH on EEG power densities during wakefulness (A), REM sleep (B) and NREM sleep (C) in rats. αs1-CH (150, 300 mg/kg) was orally administrated to rats once daily for 3 days. Power densities were classified as δ-wave, θ-wave, and α-wave densities. The Newman-Keuls test was used to compare groups versus the naïve control group. Columns represent mean values and SEMs (n=5–6). p-values of <0.05 or <0.01 were considered significant (as indicated by * or **, respectively).

Fig. 5. Modulation of glutamic acid decarboxylate (GAD) and GABAA receptor subunits in the rat hypothalamus by αs1-CH (150, 300 mg/kg). After three days of consecutive treatment by αs1-CH, hypothalamic tissues were isolated, homogenized, and immunoblotted. Immunoreactive band intensities were measured by densitometry. Protein levels were normalized versus GAPDH. Columns contain mean values and SEMs (n=3). The Newman-Keuls test was used to compare groups versus the naïve control group. *p-values of < 0.05 were considered significant.